Jian et al BMC Genomics (2020) 21:343 https://doi.org/10.1186/s12864-020-6716-8 RESEARCH ARTICLE Open Access Differential expression pattern of the proteome in response to cadmium stress based on proteomics analysis of wheat roots Mingyang Jian1†, Dazhong Zhang1†, Xiaoying Wang1, Shuwei Wei1, Yue Zhao1, Qin Ding2*, Yucui Han1* and Lingjian Ma1* Abstract Background: Heavy metal cadmium (Cd) is a common environmental pollutant in soils, which has an negative impacts on crop growth and development At present, cadmium has become a major soil and water heavy metal pollutant, which not only causes permanent and irreversible health problems for humans, but also causes a significant reduction in crop yields Results: This study examined the chemical forms of Cd in the roots of two wheat varieties (M1019 and Xinong20) by continuous extraction and analyzed differences in distribution characteristics of Cd in the root cell wall, cytoplasm, and organelles by elemental content determination and subcellular separation Furthermore, we conducted proteomics analysis of the roots of the two varieties under Cd pollution using mass spectrometry quantitative proteomics techniques A total of 11,651 proteins were identified, of which 10,532 proteins contained quantitative information In addition, the differentially expressed proteins in the two varieties were related to DNA replication and repair, protein metabolism, and the glutathione metabolism pathway Conclusion: The results of this study improve our understanding of the mechanism of plant responses to Cd stress Keywords: Cd stress, Wheat roots, Chemical forms, Proteomics analysis Background The harmful effects of nonliving factors on organisms in a specific environment are called abiotic stress [1] In the natural environment, abiotic stresses including heat, cold, and heavy metals are not only harmful to the environment, but also detrimental to plants and agriculture [2, 3] Recent studies have shown that environmental pollution, such as heavy metal pollution, poses a serious threat to living organisms Plants can cause dysfunction of plant * Correspondence: dingqin@nwsuaf.edu.cn; 455205716@qq.com; malingjian@nwsuaf.edu.cn † Mingyang Jian and Dazhong Zhang contributed equally to this work College of Horticulture, Northwest A&F University, Yangling 712100, China College of Agronomy, Northwest A&F University, Yangling 712100, China proteins under heavy metal stress [4] With the intensification of human activities such as mining and industrial activities and the excessive using of phosphate fertilizer, cadmium is released into the natural environment through geological and human activities Even at low concentrations, cadmium is one of the most toxic elements, which is due to high fluidity and bioavailability Therefore, cadmium can more easily be absorbed in the underground part of plants and accumulated in the aerial part [5, 6] Wheat is one of the most important and the foremost food crops in China In 2011–2012, wheat production was significantly higher than corn and rice (USDA 2011) Globally, the main food source for human protein intake is wheat, which © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data Jian et al BMC Genomics (2020) 21:343 has higher protein content than that of corn and rice [7, 8] Cd is a toxic heavy metal that is absorbed by the roots, subsequently inhibiting plant growth and development Roots are the first organs that are exposed to Cd ions Studies have shown that the first defense strategy is to exclude the metal entering the root tissues Roots rapidly respond to the presence of Cd by forming a mechanical barrier [9, 10] Cadmium, absorbed by roots and accumulated in plants, may lead to physiological, biochemical and structural changes in plants such as free radicals, which directly cause cell peroxidation It would influence photosynthetic and stimulates leaf apoptosis and necrosis [11–13] In order to respond cell peroxidation, cadmium can be detoxified by phytochelatin (a glutathione-derived peptide) or use antioxidant enzymes to degrade excess reactive oxygen species (ROS) [14, 15] In conclusion, plants adapt to adverse conditions through a series of physiological, cellular, and molecular processes, culminating in stress tolerance [16] Extensive studies have been conducted on the physiological aspects of plant stress responses, whereas proteomics investigations, particularly involving wheat roots, are limited In order to better explain the resistance mechanism of wheat stress on cadmium, proteomics as a useful analysis tools have been used to analyze protein changes due to changes in environmental factors Proteins are the end products of genes and play an important role in plant cell metabolism and biological processes With the deepening of proteomics research, they have made an important supplement to gene expression research Page of 13 (Fig 1) The proportion of Cd in different chemical forms of M1019 variety was sodium chloride extraction state > acetic acid > extraction state > deionized water extraction state > ethanol extraction state> hydrochloric acid extraction state The proportion of Cd in different chemical forms of Xinong20 variety is sodium chloride extraction state > deionized water extraction state > ethanol extraction state > acetic acid extraction state > hydrochloric acid extraction state From the content of Cd of subcellular component (Fig 2), Cd in root tissues of M1019 and Xinong20 was mainly distributed in the cell wall, however the Cd content of M1019 was the lowest in organelles, and the Cd content of Xinong20 was lowest in cytoplasmic The original statement has been changed: The content of cadmium in the cell wall of M1019 was higher than that of xinong20, and the content of cadmium in the cell fluid and cytoplasm was lower than that of xinong20 In addition, we used wolfpsort, a subcellular localization prediction software, to predict the subcellular localization The results showed that the upregulation protein that located in cytoplasm of M1019 is a little more than Xinong20 However, the downregulated M1019 located in cytoplasm was approximately half as much as Xinong20 (Figs and 4) The cytoplasm is the main site for biochemical reactions; therefore, Cd stresses may imparted greater effects on Xinong20 To further analyze the differentially expressed proteins, we conducted systematic bioinformatics analysis Analysis of mass spectrometry data Results Distribution of cadmium and DEPs subcellular structure localization The combined form of M1019 and Xinong20 Cd is mainly based on the extracted state of sodium chloride We conducted quality control testing of the obtained mass spectrometry data First, mass errors in all identified peptides were assessed The results showed that the quality error was centered at and concentrated to a range below 10 PPM, indicating that the quality error Fig Cadmium chemical analysis After t-test, the significant differences at the level of 0.01 Jian et al BMC Genomics (2020) 21:343 Page of 13 Fig Distribution of cadmium in the cell wall, organelle components, and cytoplasmic After t-test, the significant difference at the level of 0.01 met the requirements Second, most of the peptide lengths were distributed between and 16 amino acid residues (Fig 5) The results showed that the sample conformed to the rule of and the sample preparation reached criterion, so the sample can be used for next step of biological information analysis Pearson’s correlation coefficient was used to determine whether the biological repetition rate was statistically consistent In this experiment, all sample pairs were used to calculate our Pearson’s correlation coefficient The closer Pearson’s coefficient is a measure of the linear correlation between sets of data When the coefficient closer to − is a negative correlation, the closer to is a positive correlation, the closer to is relevant The results (Fig 6) showed Fig DAPs subcellular structure localization of M1019 that the three biological repeats were statistically consistent, indicating that the results were reliable A total of 11,651 proteins were identified, of which 10, 532 contained quantitative information Taking 1.5 times as the change threshold and t-test p-value< 0.05 as the standard, we determined that the expression of 268 proteins in M1019/M1019ck was upregulated and 217 proteins were downregulated The expression of 684 proteins in Xinong20/Xinong20ck was upregulated, and that of 680 proteins was downregulated Gene ontology enrichment analyses of DEPs We conducted GO annotation analysis on the identified differential proteins and explained the biological effects Jian et al BMC Genomics (2020) 21:343 Page of 13 Fig DAP subcellular structure localization of Xinong20 of proteins from different angles Therefore, we conducted statistical analysis of the distribution of proteins quantified by M1019 and Xinong20 in the GO secondary annotations The GO annotation is divided into three primary categories: the biological process, the cellular component, and the molecular function (Figs and 8) Among the biological process functions, the upregulated expression, the DEPs of M1019 were related to responses to stress and carbon fixation The DEPs of Xinong20 were related to decomposition and amino sugar catabolic process, the cell wall macromolecule catabolic process, polysaccharide catabolic process, regulation of protein catabolic process, and protein ubiquitination For downregulated expression, the DEPs of Fig the mas error and peptide lengths of the sample Three biological replications were performed for proteomics analysis of the samples, and the left, center and right respectively represents the result of the first, second and third replications Jian et al BMC Genomics (2020) 21:343 Page of 13 related to external encapsulating structure The DEPs of Xinong20 were related to ribosomes and ribonucleoprotein complexes (Figs and 8) Genomes (KEGG) enrichment analysis of DEPs Fig Heat map draw for calculating our Pearson’s Correlation Coefficient using all sample pairs The A represents wheat M1019, and A1, A2, and A3 represent the three biological replications of cadmium treatment Ack1, Ack2 and Ack3 represent three biological replications of the control (nutrient solution only without cadmium) The B represents wheat xinong20 and B1, B2 and B3 represent three biological replications of cadmium treatment Bck1, Bck2 and Bck3 represent three biological replications of the control group (nutrient solution only without cadmium) M1019 were related to cellular catabolic process The differentially expressed proteins of Xinong20 were mainly related to the balance of the ion, cell metabolism, and macromolecular biosynthesis Destruction of ion balance may be a factor that Xinong20 is sensitive to Cd stress Among the molecular process functions, the upregulated expression, the DEPs of M1019 were related to transferase activity, transferring glycosyl groups, and metal ion binding The differentially expressed proteins of Xinong20 were mainly related to peptidase inhibitor activity, including those of endopeptidases Inversely, the helicase activity of DNA and hydrolase activity, which act on ester bonds, enzyme inhibitor activity, and peptidase inhibitor activity were downregulated Therefore, the difference in inhibiting enzyme activity will affect peptide synthesis in Xinong20 to some extent, which may indirectly affect protein metabolism process Among the cellular component process functions, the upregulated expression, the DEPs of M1019 were related to intrinsic and integral components of the membrane The DEPs of Xinong20 were related to the extracellular region For the downregulated expression, the DEPs of M1019 were We also made comparisons with the KEGG database to predict possibly important pathways For upregulated expression, both M1019 and Xinong20 enriched the MAPK signaling pathway-plant and glutathione metabolism However, the results showed that most of the DEPs in the MAPK signaling pathway of Xinong20 were related to pathogenic proteins A significantly greater enrichment of the glutathione metabolism pathway of M1019 than that in Xinong20 was observed (Fig 9) For the upregulated expression, the results indicated that the DEPs of M1019 also enriched in protein processing in endoplasmic reticulum, alpha-linolenic acid metabolism, phenylalanine metabolism, galactose metabolism, and cysteine and methionine metabolism (Table 1) In terms of downregulated expression, more pathways in Xinong20 were enriched than that observed in M1019 The DEPs related to glycosphingolipid biosynthesis-globo and isoglobo series were highly enriched in M1019 and Xinong20 Moreover, the DEPs related to nitrogen metabolism were enriched in M1019 such as the glutamine synthetase cytosolic isozyme The DEPs were related to DNA replication, homologous recombination, mismatch repair, and ribosomes Lysine biosynthesis, nitrogen metabolism, and protein export were also enriched in Xinong20 In terms of protein export pathways, the DEPs in Xinong20 were associated with transport proteins such as the mitochondrial inner membrane protein and signal recognition particle 54 kDa For the ribosome pathway, numerous DEPs were enriched in Xinong20 In addition, the DNA polymerase delta small subunit and replication protein 32 kDa subunit showed downregulated in the DNA replication, mismatch repair, and homologous recombination pathway (Table 2) Discussion Cd content distribution and chemical form distribution Most of the heavy metal absorption takes place through root cells, which distribute toxic ions to specific organs, tissues, or organelles Studies have shown that metal ions in Ni-rich plant cells, Zn hyperaccumulators and copper hyperaccumulators are mainly distributed on the cell wall, and the content in organelles is relatively small [17, 18] In this study, the cadmium distribution of M1019 in root cells was the same as that in Ni-rich plant cells, Zn hyperaccumulators and copper hyperaccumulators, while the content of cadmium in cytoplasmic component of Xinong20 was higher than that in organelles Therefore, M1019 can better accumulate cadmium in cell wall, Jian et al BMC Genomics (2020) 21:343 Fig Gene Ontology (GO) analysis of the upregulation of M1019 and Xinong20 Fig Gene Ontology (GO) analysis of downregulation of M1019 and Xinong20 Page of 13 Jian et al BMC Genomics (2020) 21:343 Page of 13 Fig Main DAPs enriched in the pathways which may be one of the factors of resistance to cadmium in M1019 In addition, the Cd content and percentage of cell organelles of M1019 were lower than Xinong20, and the Cd content and percentage of cell wall were higher than Xinong20 Therefore, M1019 can more effectively accumulate Cd in the cell wall and prevent excess Cd from entering cellular organelles According to the GO analysis, among the cellular component process, for the downregulated expression, the DEPs were detected in cytoplasmic part So Xinong20 may be more effected than M1019 Studies have shown that absorption operations play an important role in maintaining trace amounts of cellular metal ions Heavy metals can combine with polar compounds to form water-soluble, alcohol-soluble, and acid-soluble low-molecular weight metal chelates to affect heavy metals in plants [19] Some studies indicate that the ethanol extraction state and water-soluble Cd have the strongest migration activity, and the acid- soluble (acetic acid and hydrochloric acid extraction) Cd had the weakest migration activity, so the Cd toxicity effect in the alcohol-soluble and water-soluble form is more significant than other chemical forms on Cd migration activity [20] In this study, the content of Cd in the ethanol-extracted and water-extracted state of M1019 variety was lower, and the content of acidsoluble Cd with weaker migration activity was higher than Xinong20 Acidic extraction with weaker migration activity may also be more conducive to cadmium deposition on the cell wall, thereby slowing down Cd stress However, the Cd with stronger migration activity is more likely to move upwards in Xinong20, thus causing toxicity to the plants On the other hand, this may contribute to the accumulation of Cd in Xinong20 plants We preliminarily concluded that the migration of Cd in M1019 is weaker than that in Xinong20, which may be a key factor for more cell wall deposition in M1019 ... changed: The content of cadmium in the cell wall of M1019 was higher than that of xinong20, and the content of cadmium in the cell fluid and cytoplasm was lower than that of xinong20 In addition,... Among the biological process functions, the upregulated expression, the DEPs of M1019 were related to responses to stress and carbon fixation The DEPs of Xinong20 were related to decomposition and... process Among the cellular component process functions, the upregulated expression, the DEPs of M1019 were related to intrinsic and integral components of the membrane The DEPs of Xinong20 were